[關(guān)鍵詞]
[摘要]
目的 基于二維液相色譜-高分辨串聯(lián)質(zhì)譜(2D-LC-HR- MS/MS)法分析頭孢丙烯原料藥雜質(zhì)譜。方法 一維色譜條件進(jìn)行樣品色譜圖采集,確認(rèn)各雜質(zhì)的出峰位置,采用 YMC Hydrosphere 色譜柱(250 mm×4.6 mm,5 μm),以 11.5 g·L-1磷酸二氫銨(用磷酸調(diào)節(jié) pH 為 4.4)為流動(dòng)相 A,乙腈-流動(dòng)相 A(50∶50)為流動(dòng)項(xiàng) B,梯度洗脫;柱溫 40 ℃;體積流量1.0 mL·min-1;檢測(cè)波長(zhǎng) 230 nm;進(jìn)樣量 4 μL。二維液相脫鹽后切進(jìn)高分辨串聯(lián)質(zhì)譜進(jìn)行分析,根據(jù)結(jié)果推斷雜質(zhì)結(jié)構(gòu)及生成機(jī)制,采用 Waters BEH C18色譜柱(50 mm×2.1 mm,1.7 μm),以 0.01% 甲酸水溶液為流動(dòng)相 A,乙腈為流動(dòng)相 B,切峰后開始 A 相由 98% 到 1%,柱溫 40 ℃,體積流量 0.3 mL·min-1,質(zhì)譜采用 Xevo G2-XS QTof MS 系統(tǒng),離子源為 ESI 源,毛細(xì)管電壓 3.0 kV,霧化器溫度 450 ℃,掃描范圍 m/z 100~2 000。結(jié)果 頭孢丙烯樣品中存在 9 個(gè)雜質(zhì)色譜峰,其中 5 個(gè)雜質(zhì)為已知雜質(zhì),峰 3 為雜質(zhì) B(頭孢羥氨芐)、峰 5 為雜質(zhì) D、峰 6 為雜質(zhì) F、峰 7 為雜質(zhì) G、峰 9 為雜質(zhì) I;對(duì)其中 3 個(gè)未知雜質(zhì)可能的結(jié)構(gòu)式進(jìn)行了初步推測(cè)以及探討了可能的生成途徑,峰 2 分子式為 C18H19N3O6S,該化合物比頭孢丙烯多一個(gè)氧,分析其為頭孢丙烯的氧化雜質(zhì);峰 4 分子式為 C16H15N3O6S,與雜質(zhì) B 相比增加了 1 個(gè)氧原子,減少了 2 個(gè)氫原子,判斷其為 7-ACA 內(nèi)酯與對(duì)羥基苯甲甘氨酸甲酯(HPGM)反應(yīng)產(chǎn)物中的硫原子繼續(xù)發(fā)生了氧化生成的;峰 8 分子式為 C8H9NO2S,該組分為頭孢丙烯分子結(jié)構(gòu)的一部分。峰 1 有待進(jìn)一步研究。結(jié)論 該方法有效解決了頭孢丙烯流動(dòng)相中含不揮發(fā)性磷酸鹽的色譜體系與色譜-質(zhì)譜快速鑒定雜質(zhì)不兼容的難題,可以簡(jiǎn)單、快速地對(duì)頭孢丙烯有關(guān)物質(zhì)進(jìn)行定性分析及雜質(zhì)譜研究。
[Key word]
[Abstract]
Objective To analyze the impurity profile of cefprozil API using 2D-LC-HR-MS/MS. Methods The sample chromatogram was acquired by one-dimensional chromatography conditions to confirm the peak positions of each impurity. The YMC Hydrosphere column (250 mm×4.6 mm, 5 μm) was used, with 11.5 g·L-1 ammonium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid) as solvent A, a mixture of acetonitrile and solvent A (50:50) as solvent B, and gradient elution; column temperature was 40℃; flow rate was 1.0 mL·min-1; detection wavelength was 230 nm; injection volume was 4 μL. The impurity profile was analyzed by 2D-LC desalting and high-resolution tandem mass spectrometry (HR-MS/MS) after cutting into the mass spectrometer. Based on the results, the structures and formation mechanisms of the impurities were inferred. The Waters BEH C18 column (50 mm×2.1 mm, 1.7 μm) was used, with 0.01% formic acid aqueous solution as solvent A and acetonitrile as solvent B. The peaks were cut and the A phase was gradually increased from 98% to 1% starting from the cut. The column temperature was 40° C, the flow rate was 0.3 mL·min-1, and the mass spectrometer used was the Waters Xevo G2-XS QTof MS system with an ESI ion source, a capillary voltage of 3 kV, a vaporizer temperature of 450 ℃, and a scanning range of m/z 100—2 000. Results Nine impurity peaks were identified in the cefprozil sample, of which five were known impurities. Peak 3 was impurity B (cefhydroxime), peak 5 was impurity D, peak 6 was impurity F, peak 7 was impurity G, and peak 9 was impurity I. Preliminary structural hypotheses were made for the three unknown impurities and the possible generation routes were discussed. Peak 2 has a molecular formula of C18H19N3O6S, which was one oxygen atom more than cefprozil. It was analyzed as an oxidation impurity of cefprozil. Peak 4 has a molecular formula of C16H15N3O6S, which was one oxygen atom and two hydrogen atoms less than impurity B. It was judged that it was the sulfur atom of the reaction product between 7-ACA lactam and HPGM (hydroxyphenylglycine methyl ester) continuing to undergo oxidation. Peak 8 has a molecular formula of C8H9NO2S, which is part of the molecular structure of cefprozil. Peak 1 needs further study. Conclusion This method effectively solved the problem of the chromatographic system with non-volatile phosphate in the mobile phase of cefprozil flowing phase and the incompatibility of chromatography-mass spectrometry rapid identification of impurities. It can simply and quickly determine the qualitative analysis and impurity spectrum of cefprozil-related substances.
[中圖分類號(hào)]
O65
[基金項(xiàng)目]
廣東省重點(diǎn)領(lǐng)域研發(fā)計(jì)劃項(xiàng)目(2022B1111070004);國(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目(61633006)